3276 MOLECULAR MEDICINE REPORTS 20: 3276-3284, 2019 Impact of SLC20A1 on the Wnt/β‑catenin signaling pathway in somatotroph adenomas JIANHUA LI1,2, WEI DONG3, ZHENYE LI4, HONGYUN WANG1, HUA GAO1 and YAZHUO ZHANG1,4 1Key Laboratory of Central Nervous System Injury Research, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100070; 2Department of Neurosurgery, Binzhou People's Hospital, Binzhou, Shandong 256610; 3Department of Neurosurgery, Tangshan People's Hospital, Tangshan, Hebei 063001; 4Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, P.R. China Received February 5, 2019; Accepted July 12, 2019 DOI: 10.3892/mmr.2019.10555 Abstract. Studies have revealed that genetic and functional was 86.4±5.7, 75.6±4.4 and 67.5±3.8%, respectively (P<0.05). aberrations of oncogenes, tumor-suppressor genes, signaling ELISA analysis demonstrated the growth hormone (GH) pathways and receptors are among the most prominent events levels in the Sh-B-SLC20A1 and Sh‑C‑SLC20A1 groups to be in pituitary tumorigenesis, and a potent biomarker would 34.7±10.4 and 54.6±14.4%, respectively, of that of control GH3 be helpful for early diagnosis, subsequent treatment and cells (P<0.05). The transmembrane invasion assay revealed that disease control. The present study investigated the expression knocking down SLC20A1 significantly suppressed cell invasion signatures of solute carrier family 20 member 1, also known in the Sh-B-SLC20A1 and Sh‑C‑SLC20A1 groups. RT-qPCR as phosphate transporter 1 (SLC20A1) and the Wnt/β-catenin and western blotting demonstrated that Sh-B-SLC20A1 and signaling pathway in 52 patients with somatotroph adenomas. Sh‑C‑SLC20A1 evidently increased the levels of Wif1 and According to immunohistochemistry analysis, the H-score secreted frizzled-related protein 4. The present data suggested of SLC20A1 was 222.6±15.2 in invasive tumor samples and that SLC20A1 levels are positively associated with tumor 144.5±30.4 in non-invasive tumor samples (P<0.01), while the size, invasive behavior and tumor recurrence in somatotroph H-scores of β-catenin were 210.1±21.4 and 134.9±32.7, respec- adenomas. Furthermore, SLC20A1 may be associated with the tively (P<0.05). The H-scores of Wnt inhibitory factor 1 (Wif1) activation of the Wnt/β-catenin signaling pathway. exhibited the opposite trend, with scores of 134.5±22.7 and 253.6±14.8, respectively (P<0.01). The H-scores of SLC20A1 Introduction were negatively associated with those of Wif1 in somato- troph adenomas (correlation coefficient r=‑0.367). The mean Somatotroph adenomas cause disfiguring growths and progression-free survival in the low SLC20A1 group was longer medical complications, including cardiovascular and pulmo- than that in the group with high SLC20A1 H‑scores (P=0.024). nary complications, and increased mortality rates due to Reverse transcription-quantitative PCR (RT-qPCR) and western malignancy (1). Acromegaly is mainly caused by somato- blotting confirmed the interference efficiency of the segments troph adenomas that secrete excess growth hormone (GH) short hairpin (Sh)-B-SLC20A1 and Sh‑C‑SLC20A1. Cell and insulin-like growth factor 1 (IGF-1) (2). Somatotroph proliferation experiments revealed that the cell viability of the adenomas are initially treated surgically, followed by radia- Sh-B-SLC20A1 group was 76.3±4.5, 65.7±3.7 and 53.1±3.2% of tion and/or medication, if necessary (3). Large tumor sizes and that of control GH3 cells after 24, 48 and 72 h of transfection, high post-operative IGF-1 levels are predictors for incomplete respectively, while the cell viability of the Sh‑C‑SLC20A1 group remission and uncontrolled disease (4). In a complex multistep process, genetic and epigenetic factors, hormonal stimulation, growth factors and their receptors contribute to tumorigenesis and the development of somatotroph adenomas. To date, no high-frequency somatic genetic alterations have been identi- Correspondence to: Dr Hua Gao or Dr Yazhuo Zhang, Key fied in somatotroph adenomas, and the somatic landscape Laboratory of Central Nervous System Injury Research, Beijing shows Ca+2 and the ATP signaling pathways to be involved in Neurosurgical Institute, Capital Medical University, 119 Southwest pituitary tumorigenesis (5). However, there is a lack of clinical 4th Ring, Beijing 100070, P.R. China evidence of phenotype-genotype correlations between the E‑mail: [email protected] E‑mail: [email protected] α subunit of stimulatory G-protein (GNAS) in patients with mutated and non-mutated somatotroph adenomas (6). Key words: somatotroph adenomas, solute carrier family 20 Inorganic phosphate (Pi) enters the cells via Na/Pi cotrans- (phosphate transporter), member 1, Wnt/β-catenin signaling pathway, porters, which comprise solute carrier (SLC)20 and SLC34 in Wnt inhibitory factor 1, invasion mammals (7). The SLC20 family consists of two members, namely SLC20A1 [also known as phosphate transporter 1 (PiT-1)] and SLC20A2 (PiT2). SLC20A1 encodes sodium-dependent LI et al: SLC20A1 IN SOMATOTROPH ADENOMAS 3277 phosphate transporter 1, whose gene is located on chromosome described method (15). The following primary antibodies were 2q11-14, a log 10 odds peak score region (8). Overexpression of used: Anti-SLC20A1 (cat. no. ab237527; 1:1,000; Abcam), SLC20A1 increases cell proliferation and colony formation in anti-β-catenin (cat. no. ab32572; 1:2,000; Abcam), anti-Wif1 murine fibroblastic NIH3T3 and pre‑osteoblastic MC3T3‑E1 (cat. no. ab155101; 1:500; Abcam) and anti-Ki 67 (cat. no. ACK02; cells. Furthermore, non-proliferating cells exhibit the lowest 1:1,000; Leica Microsystems, Inc.). A secondary antibody SLC20A1 mRNA levels in these cell lines (9). Overexpression (cat. no. sc-2040; 1:5,000; Santa Cruz Biotechnology, Inc.) was of SLC20A1 leads to faster adhesion and spreading compared used in combination with the Bond Polymer Refine Detection with control NIH3T3 cells (10). High SLC20A1 expression was system (cat. no. DS9800; Leica Microsystems, Inc.). Staining associated with worse survival in 401 patients with estrogen intensity was stratified on a scale of 0‑3 as follows: 0, no staining; receptor-positive breast tumors compared with low SLC20A1 1, weak staining; 2, moderate staining; and 3, strong staining. The expression (10-year survival rates, 17.16 vs. 68.45%) (11). The H-score was obtained by multiplying the staining intensity by a epidermal growth factor receptor inhibitor gefitinib exhibits constant to adjust the mean to the strongest staining, to produce strong inhibition of cell proliferation in glioblastoma cell lines a score in the range of 0‑300. Specifically, H‑score=scale x by regulating SLC20A1 expression (12). In addition, SLC20A1 percentage of strong staining (0‑100%). H‑score=1.0 indicated a modulates erythroid maturation by modulating the activity of weak percentage; 2.0 indicated a moderate percentage; and 3.0 erythroid Kruppel-like factor in vivo and in vitro (7). indicated a strong percentage. The Wnt signaling pathway has been implicated in the processes of tissue differentiation, proliferation, apoptosis Cell culture, proliferation and migration. GH3 cells were and tumorigenesis (12). The levels of Wnt inhibitory factor 1 purchased from the American Type Culture Collection (ATCC) (Wif1) and secreted frizzled-related protein 4 (sFRP4) are and were cultured in F12K (ATCC) supplemented with 2.5% lower in invasive nonfunctional pituitary adenomas (NFPAs) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and than in noninvasive NFPAs (13). The present study investigated 15% horse serum (Gibco; Thermo Fisher Scientific, Inc.) under the expression signatures of SLC20A1 and the Wnt/β-catenin 37˚C and 5% CO2. signaling pathway in 52 patients with somatotroph adenomas. The GH3 cells were plated onto 96-well dishes with Furthermore, it explored the possibility that SLC20A1 could 1x104 cells and 100 µl medium in each well, and incubated be targeted by chemotherapeutic agents or used as a molecular overnight. SLC20A1 gene silencing in the GH3 cells was marker for the diagnosis of patients with cancer via RNA established by transfection of short hairpin RNA (1 µg for interference (RNAi) technology in the GH3 cell line. 1x106 cells; OriGene Technologies, Inc.) or non-targeting control shRNA (scramble) for 72 h using 5 µl Opti-MEM Materials and methods (Gibco; Thermo Fisher Scientific, Inc.) and 0.3 µl Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Patients and tissue specimens. The present study retro- Inc.), according to the manufacturer's instructions. The spectively reviewed 52 patients (mean age, 40.3 years; range sequences of the pGFP‑C‑shLenti plasmid were: Scramble, 16-62 years) with somatotroph adenomas at Beijing Tiantan TTC​TCC GAA​CGT GTC​ACG T; A, CAC​CGA​AGT ATG​ACA​ Hospital affiliated to Capital Medical University, between ATC​TGT GGA​TGC​TC; B, GCA​ATG CTG TGT CTG​ACC​ July 2012 and December 2016. The diagnosis of somatotroph TTC​ACT​CAG AG; C, ATA​GGA​ATC​CTG TGT CTG AGG adenoma was based on clinical symptoms and histopatho- TAG TAT GT; and D, GCT TCT GCT​CCA​CCG​AAG TAT GAC​ logical features, per the 2017 World Health Organization AAT​CT. Then, 20 µl MTS solution was added to each well Classification (14). All patients were diagnosed with sporadic and further incubated for 4 h. The absorbance at 490 nm of somatotroph adenomas. The inclusion criteria for recruitment each well was measured using an ELISA plate reader (Thermo of patients were as follows: i) Serum GH >1 ng/ml and IGF-1 Fisher Scientific, Inc.). above normal (115-307 ng/ml), or GH >2 ng/ml; ii) magnetic Cell migration and invasion were measured using fibro- resonance imaging confirmed the existence of the saddle area; nectin‑ and Matrigel‑coated polycarbonate filters, respectively, iii) acromegaly or facial changes; and iv) transmission electron and modified transwell chambers (Corning Inc.). In total, microscopy revealed large secretory granules (400-600 nm).